1. Significance of Non-Standard Peptides
Many drugs currently on the market are low-molecular-weight organic compounds having a molecular weight of 500 Da or lower. Low-molecular-weight compounds are used because they are quickly absorbed and dispersed in the body, they often exhibit good cell membrane permeability, and are often not immunogenic. However, possible side effects amongst these molecules often result from their low selectivity. In contrast, the recently popular antibody drugs exhibit high selectivity, but they can be immunogenic, and their targets are basically limited to extracellular ones or those on cell surfaces, so their application as pharmaceutical agents is relatively limited.
Meanwhile, non-standard peptides are considered to be a group of molecules that combine the advantages of both low-molecular-weight compounds and antibodies. Developing peptide drugs has proven challenging, since peptides often exhibit low membrane permeability due to their molecular weight (1,000 to 3,000 Da) being higher than that of a low-molecular-weight organic compound, and because peptides are often rapidly degraded in vivo by proteases, thus exhibiting poor pharmacokinetic properties. However, cyclosporine, which is a naturally derived peptide, permeates cell membranes and exhibits immunosuppressive effects by binding to its target. These biological activities and pharmacokinetics result from enhanced target-binding strength, membrane permeability, and in vivo stability caused by the macrocyclic structure and the special (non-standard) amino acids in the peptide. Hence, a non-standard peptide is potentially capable of targeting not just extracellular targets but also intracellular targets. Meanwhile, significant effort has been put into developing therapeutics to inhibit defective interactions, since many diseases/disorders originate from defects in the interaction between proteins. It is normally difficult to develop inhibitors to these protein-protein interactions based on low-molecular-weight compounds, since the proteins often bind to each other via a wide area (750 to 1,500 Å) with no clear hydrophobic pockets. In contrast, non-standard peptides per se are relatively larger and have functional groups that cause static and hydrophobic interactions and enable hydrogen bonds to form. Hence, non-standard peptides could potentially bind to shallow and wide/flat binding sites on proteins in an effective manner. Thus, non-standard peptides, which are capable of binding to a wide range of extracellular/intracellular protein targets, represent an extremely attractive group of potentially new drugseeds.
2. Effects of Macrocyclic Structure and N-Methyl Amino Acid on Peptide
The macrocyclic structure provides peptides with advantages as pharmaceutical agents. 1) The macrocyclic structure limits the conformational space of the molecule and thus reduces the entropy loss when binding with the target; it thus provides the molecule with a stronger binding capacity than a straight chain structure. It has also been reported that target selectivity improves through cyclization. 2) In vivo proteases break/degrade natural polypeptides, so it binds to and cleaves straight/linear chain peptides. However, this ability of proteases to degrade cyclic peptides is much lower, and therefore in vivo stability of cyclic peptides is considerably improved. 3) It is also understood that the rigid structure improves membrane permeability. This is due to the peptide cyclization increasing the number of amide bonds forming hydrogen bonds in the molecule, and decreasing the energy loss during the desolvation of amide N—H under a hydrophobic environment in the cell membrane.
Meanwhile, the use of N-methyl amino acid result in the formation of an N-methyl peptide bond to be incorporated into the backbone of the peptide chain. 1) This structure, similar to a cyclic structure, is not easily identified or cleaved by proteases. 2) As an example, the incorporation of N-methyl peptide bonds improved cell membrane permeability and intestinal absorption resulting in increased bioavailability. 3) The double bond in a peptide bond allows the peptide bond to take both the cis-structure and the trans-structure, but the trans-structure is taken in conventional peptide bonds to avoid the high allylic strain of the cis-structure. However, the difference between the allylic strain of the cis-structure and that of the trans-structure in the N-methyl peptide bond is lower than that of conventional peptide bonds, so it occasionally takes a cis-structure. Thus, the structure of the entire peptide may change considerably by the insertion of one or more N-methyl peptide bonds to form a special structure that is not possible in normal peptides. It has thus been suggested that peptides that contain N-methyl peptide bonds can prove to be an effective library capable of binding to target protein surfaces, against which conventional peptides cannot provide a sufficient binding.
3. Methods for Synthesizing N-Methyl Peptides Using a Translation System, Constructing a Library of N-Methyl Peptides, and Searching for a Pharmaceutical Candidate Using Such Libraries
Naturally-derived N-methyl peptides are synthesized by enzyme groups called non-ribosomal peptide synthetases (NRPS). These enzyme groups are extremely complicated, and no technology is currently established to create a peptide library by artificially tailoring these enzymes.
Several synthesis methods of N-methyl peptides that use translation systems to enable limit-free creation of N-methyl peptides have been reported thus far. Summarized below are studies on the N-methyl peptide translation synthesis that uses altered genetic codes created through artificially reassigning natural amino acids with N-methyl amino acids.
N-methylphenylalanine (Bain et al., Tehtahedron, 1991, 47, 2389-2400. Rabbit reticulocyte lysate), N-methylalanine (Ellman et al., Science, 1992, 255, 197-200. E. coli lysate), N-methylglycine (Chaung et al., Science, 1993, 259, 806-809. E. coli lysate), N-methyl aspartic acid (Karginov et al. JACS, 1997, 119, 8166-8176. Short et al., Biochemistry, 2000, 39, 8768-8781. Rabbit reticulocyte lysate) have been reported to be incorporated into one position of a peptide/protein using a UAG (terminator) codon.
In addition, a similar method using a sense codon have also been reported. Many such examples use a reconstituted cell-free translation system (reconstituted cell-free translation system; Y. Shimizu et al., Nature Biotechnology, 2001, vol. 19, p. 751-755, etc.) as a peptide expression system to avoid competition with natural amino acids.
Green et al. performed translation-synthesis of dipeptides using 20 types of N-methyl amino acids (Merryman et al., Chem. Biol., 2004, 11, 575-582. Reconstituted in vitro translation system. E. coli). The N-methyl aminoacyl tRNA was prepared according to the following three steps: 1) protecting the amino group with 2-nitrobenzaldehyde using aminoacyl-tRNA provided by the aminoacyl tRNA synthetase (ARS) as the substrate; 2) methylating the amino group with formaldehyde; 3) deprotecting 2-nitrobenzyl group by UV radiation. Szostak et al. successfully synthesized N-methyl peptides consisting of the following three N-methyl amino acids: N-methylvaline, N-methylleucine, N-methylthreonine (Subtelny et al., JACS, 2008, 130, 6131-6136, Reconstituted in vitro translation system. E. coli).
Cornish et al. successfully translationlly-synthesized a tripeptide by assigning N-methylalanine or N-methylphenylalanine to the GUU codon (valine) (Tan et al., JACS, 2004, 126, 12752-12753, Reconstituted in vitro translation system. E. coli).
Kawakami et al. successfully incorporated various N-methyl amino acids using Flexizyme, which is an RNA catalyst (ARS ribozyme) having an acyl tRNA synthetase-like activity (Kawakami et al., Chem. Biol., 2008, 15, 32-42. Reconstituted in vitro translation system. E. coli). They have successfully synthesized an N-methyl peptide comprising 10 continuous residues consisting of 3 types of N-methyl amino acids. Kawakami et al further translationally synthesized a cyclic peptide containing four N-methyl amino acids incorporated therein.
Typical ARS ribozymes are described in the following documents (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 “A highly flexible tRNA acylation method for non-natural polypeptide synthesis”; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894 “A flexizyme that selectively charges amino acids activated by a water-friendly leaving group”; and WO 2007/066627 “Multi-Purpose Acylation Catalayst and Use thereof”).
On the other hand, only one example has been published so far concerning the construction of a peptide library consisting of N-methyl amino acids using a translation system and its application, which is a search for pharmaceutical agent candidates. Roberts et al. translation-synthesized an N-methyl peptide library consisting of N-methylphenylalanine assigned to the GUA codon (valine) and confirmed that such method is compatible with the mRNA display method (Frankel et al., Chem. Biol., 2003, 10, 1043-1050. tRNA-depleted rabbit reticulocyte lysate). Further, the same group reported a peptide cyclization method using a crosslinking agent (disuccinimidyl glutarate, DSG) and a mRNA display method that assigns N-methylphenylalanine to a UAG codon in the random NNS (wherein, N is one of A, R, C or G and S is C or G) region (Millward et al., ACS Chem. Biol. 2007, 2, 625-634. Rabbit reticulocyte lysate). As a result, a cyclic peptide binding to a target protein was obtained, however no N-methylphenylalanine was found in the selected peptide. It is not clear whether such result is due to the low quality (that is, the quality assurance of whether a peptide containing N-methylphenylalanine is included in the library) of the library constructed by Roberts et al. per se, or to the absence of N-methylphenylalanine in the active peptide, but the result clearly indicates that it is difficult to obtain biologically active species of the desired non-standard peptide containing N-methyl amino acids with the current maturity level of the given technology/methods.
As the above examples indicate, no peptide library has been created so far that contains more than a single N-methyl amino acid, nor has there been any successful selection or identification of such a peptide from such library(ies).
4. Preparation of Peptide Libraries Using In Vitro Display
In vitro display is a system that displays the phenotype with the genotype through conjugating a phenotype and a genotype, which encodes the sequence of the phenotype, by a noncovalent bond or a covalent bond and enables enrichment and amplification (selection) of active species using replication systems reconstituted in test tubes. The greatest advantage of the present system is that it allows one to search through a library encompassing a wide variety of nonstandard peptides, made possible by excluding prokaryote and eukaryote organisms from use as mediums, enabling the selection of highly active physiological substance (i.e. peptide herein). A typical comparative example is that of a phage display using E. coli as the replication medium, which enables a selection from a library with a diversity of 10 to the 7th power. In comparison, an in vitro display enables one to search a library with a diversity of 10 to the 13th power. In vitro display includes ribosome display, mRNA display, PD display (patented as RAPID display). Although mRNA display is explained below as an example, the library of non-standard peptide compounds disclosed in the present specification is applicable to all in vitro displays.
mRNA display is a technology in which the peptide is linked to its template mRNA, allowing the pairing of the amino acid sequence of a peptide with its nucleic acid sequence. To achieve such a complex, a puromycin, which is a terminal analog of acylated tRNA, is linked to the 3′ terminal of mRNA via a suitable linker, and the linked product is added to the translation reaction to incorporate puromycin to site A of the ribosome and form a covalent bond of puromycin and a peptide in the process of elongation. Consequently, the translation product, that is the peptide molecule, remains conjugated to its template mRNA via the puromycin (Roberts et al., Proc. Natl. Acd. Sci. USA, 1997, 94, 12297-12302, Nemoto et al., FEBS Lett., 1997, 414, 405-408, JP 3683282 B (WO98/16636), JP 3683902 B, JP 3692542 B (WO98/31700)).
Peptide libraries having a variety of 10 to the 13th power can be prepared by such in vitro display method, but the libraries reported thus far have been constituted of proteinogenic amino acids only. There is no known example of one successfully creating a library comprising genotypes that display peptides containing multiple special (non-standard) amino acids (including N-methyl amino acid) and cyclic structures, and performing selection therefrom.
5. HPV and Uterine Cervix Cancer
Among cancers specific to women, uterine cervix cancer is second only to breast cancer in the number of occurrences. There were 470,000 occurrences and 230,000 deaths reported worldwide annually, and 10,000 or more occurrences and 3,000 or more deaths reported in Japan annually. The prerequisite of developing uterine cervix cancer is Human papillomavirus (HPV) infection, especially high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73-type) HPV infection. Then, after 10 to 30 years of latent infection, the cell goes through malignant transformation to develop uterine cervix cancer. However, uterine cervix cancer can be almost completely (100%) prevented by preventing HPV infection, and HPV vaccines, Cervarix (GlaxoSmithKline) and Gardasil (Merck), are used in at least 100 countries around the world, and approved in Japan since September 2009. Unfortunately, the vaccines are ineffective on virus carriers, and they are ineffective against HPV infections other than type 16 HPV and type 18 HPV, since the capsid proteins of those HPVs were used as antigens in the vaccines. There is thus a need for the development of a therapeutic agent to treat uterine cervix cancer.
Latent infection of HPV spreads through the replication of the HPV genome in HPV infected cells and their distribution to daughter cells. In the process, the HPV genomes are incorporated into the host genomes so that proteins encoded by the HPV initial gene group are expressed at a high level, and an intracellular environment that is advantageous to the replication of virus genomes appears. Matters considered particularly important are immortalization, growth promotion, and inactivation of the tumor suppressor gene caused by the virus proteins E6 and E7.
E7 is a protein consisting of about 100 amino acid residues, and consists of CR1 (conserved region 1) on the N terminal, CR2 and a zinc finger domain. It binds to an Rb family protein (pRb, p107, p130) via the LXCXE motif of CR2. Rb creates a complex with the transcription factor E2F, inactivates E2F and arrests the cell at the G0 phase. However, when E7 binds to Rb, E2F is freed and activated, and the cell cycle restarts. Further, E7 binds mutually to both pRb and μ-calpain to accelerate the decomposition of pRb.
E6 binds with ubiquitin ligase E6AP in the host cell via the LXXLL motif in E6 to promote the ubiquitination and decomposition of the cancer inhibitor p53. p53 arrests the cell cycle at the G1 phase, and induces recovery when the DNA is damaged and induces apoptosis when the damage is great. When E7 inactivates Rb family proteins and moves the cell cycle forward, apoptosis is induced via p53. E6 prevents apoptosis and promotes virus growth by inhibiting the above growth arrestive effects of p53. In addition, the E6-E6AP complex promotes the ubiquitination and decomposition of a protein group and a telomerase inhibitor NFX1, the protein group having a PDZ domain that is responsible for maintaining cell polarity and controlling cell growth; accordingly, the E6-E6AP complex works in various ways to induce cancer conversion/proliferation. Chromosomes of currently available uterine cervix cancer cells, namely, HeLa cells, SiHa cells, and Caski cells, respectively include HPV18, HPV16, HPV16 genomes, which induces high expression of E6. It is observed that p53 is accumulated and apoptosis is induced when E6 or E6AP is knocked down by siRNA, indicating that the effects of E6 depend on E6AP.
6. Ubiquitin Ligase E6AP
The ubiquitin ligase E6AP (E6 associated protein, 852 amino acid residues), encoded by the UBE3A gene was discovered in 1990 by its function to bind to the cancer inhibitor p53 via E6, promoting its ubiquitination and decomposition using the 26S proteasome. The C terminal domain of E6AP, consisting of about 350 amino acid residues, is called the HECT (Homologous to E6AP Carboxyl-Terminus) domain, and forms a large family of ubiquitin E3 ligases. About 50 types of ubiquitin ligases contain a HECT domain have been confirmed in human and 5 types have been confirmed in yeast. The HECT domain includes a a large N terminal lobe (about 250 amino acid residues) and a C terminal lobe (about 100 amino acid residues) linked by a short hinge section, and in the C terminal lobe, there exists an activation cysteine. The activation E2 protein that has be ubiquitinated binds to the N terminal lobe in the HECT domain to transfer the ubiquitin to the cysteine in the C terminal lobe and forms a ubiquitin thioester intermediate. Subsequently, the amino group on the lysine side chain of the target protein recruited by the domain upstream of the HECT domain forms an isopeptide bond with the ubiquitin on the HECT to ubiquitinate the target. In former studies, E6 was bound to the α-helix consisting of 18 amino acids that are upstream of the HECT domain of E6AP by about 120 amino acid residues to form an E6-E6AP complex. The complex binds with p53 to poly-ubiquitinate p53, and induces decomposition.
Thus far there are no inhibitors of ubiquitin ligase E6AP that have been investigated as therapeutic agents for the treatment of uterine cervix cancer. In addition, there are no inhibitors of ubiquitin ligase E6AP specifically targeting the HECT domain. Furthermore, it can be predicted that agents targeting human-derived E6AP could represent a novel therapeutics class against various high-risk HPV-derived uterine cervix cancers.